title: Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci.
abstract: BACKGROUND
  Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic.
  
  
  RESULTS
  A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous 'arms' target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional.
  
  
  CONCLUSIONS
  The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Suriana
authors.initials: S
authors.lastname: Sabri
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Steen
authors.forename: Mareike
authors.initials: M
authors.lastname: Bongers
authors.forename: Lars K
authors.initials: LK
authors.lastname: Nielsen
authors.forename: Claudia E
authors.initials: CE
authors.lastname: Vickers
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D059765:Homologous Recombination; D016623:Ion Pumps / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D053858:Metabolic Networks and Pathways; D009097:Multienzyme Complexes / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D016623:Ion Pumps; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; C102053:RbsA protein, E coli; C489909:RbsR protein, E coli; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D043364:Endo-1,4-beta Xylanases; C486481:ArsAB ATPase, E Coli